Download Bundle sheath suberization in grass leaves

Journal of Experimental Botany, Vol. 65, No. 13, pp. 3371–3380, 2014
doi:10.1093/jxb/eru108 Advance Access publication 22 March, 2014
Review paper
Bundle sheath suberization in grass leaves: multiple barriers
to characterization
Rachel A. Mertz1,2 and Thomas P. Brutnell2,*
1 2 Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA
Donald Danforth Plant Science Center, St Louis, MO 63132, USA
* To whom correspondence should be addressed. E-mail: [email protected]
Received 13 December 2013; Revised 14 February 2014; Accepted 17 February 2014
Abstract
High-yielding, stress-tolerant grass crops are essential to meet future food and energy demands. Efforts are underway to engineer improved varieties of the C3 cereal crop rice by introducing NADP-malic enzyme C4 photosynthesis
using maize as a model system. However, several modifications to the rice leaf vasculature are potentially necessary,
including the introduction of suberin lamellae into the bundle sheath cell walls. Suberized cell walls are ubiquitous
in the root endodermis of all grasses, and developmental similarities are apparent between endodermis and bundle
sheath cell walls. Nonetheless, there is considerable heterogeneity in sheath cell development and suberin composition both within and between grass taxa. The effect of this variation on physiological function remains ambiguous
over forty years after suberin lamellae were initially proposed to regulate solute and photoassimilate fluxes and C4 gas
exchange. Interspecies variation has confounded efforts to ascribe physiological differences specifically to the presence or absence of suberin lamellae. Thus, specific perturbation of suberization within a uniform genetic background
is needed, but, until recently, the genetic resources to manipulate suberin composition in the grasses were largely
unavailable. The recent dissection of the suberin biosynthesis pathway in model dicots and the identification of several promising candidate genes in model grasses will facilitate the characterization of the first suberin biosynthesis
genes in a monocot. Much remains to be learned about the role of bundle sheath suberization in leaf physiology, but
the stage is set for significant advances in the near future.
Key words: Bundle sheath, C4, cell wall, endodermis, grasses, mestome sheath, physiology, suberin.
Introduction
Increased yield, and stress tolerance under marginal growing
conditions are urgently needed from grass crops to keep pace
with food and biofuel needs (Ray et al., 2013). C4 grasses are
well suited to this task, as they have greater water and nitrogen-use efficiencies than their C3 counterparts, and perform
better under hot, dry conditions (Sage, 2004; Taylor et al.,
2010). C4 species comprise the most productive cereal crops
and are promising biomass feedstocks for next-generation
biofuels (Taylor et al., 2010; Byrt et al., 2011). Given the high
productivity associated with C4 plants, efforts are currently
underway to engineer C4 photosynthesis into the C3 crop rice
(Oryza sativa), with the NADP-malic enzyme (NADP-ME)
subtype serving as a prototype for engineering efforts. If successful, it is predicted that C4 rice will increase yields by 50%
Abbreviations: ABCG, ATP-binding cassette subfamily G; ASFT, aliphatic suberin feruloyl transferase; BS, bundle sheath; CC, companion cell; CS, Casparian strips;
DCA, dicarboxylic acid; di-OH, dihydroxy (fatty acid); ER, endoplasmic reticulum; ECR, enoyl-CoA reductase; FAE, fatty acid elongase; GDSL, GDSL-like lipase/
acylhydrolase; G3P, glycerol-3-phosphate; HACD, hydroxyacyl-CoA dehydrase; ITW, inner tangential wall; KCS, ketoacyl-CoA synthase; KCR, ketoacyl-CoA reductase; LACS, long chain acyl-CoA synthetase, LCFA, long chain fatty acid; M, mesophyll; 2-MAG, sn-2 monoacylglycerol; MS, mestome sheath; MX, metaxylem
vessel; NAD-ME, NAD-malic enzyme; NADP-ME, NADP-malic enzyme; ω-OH, omega-hydroxy (fatty acid); OTW, outer tangential wall; PCK, phosphoenolpyruvate
carboxykinase; PCW, primary cell wall; PD, plasmodesmata; PM, plasma membrane; RW, radial wall; SL, suberin lamellae; ST, sieve tube; Suc, sucrose; TCW,
tertiary cell wall; VLCFA, very long chain fatty acid; VP, vascular parenchyma.
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3372 | Mertz and Brutnell
and require significantly less fertilizer than existing varieties
(Hibberd et al., 2008; Sage and Zhu, 2011). However, successful engineering of NADP-ME C4 photosynthesis into
rice may require a suite of anatomical modifications, including increased vein density, a photosynthetic bundle sheath
(reviewed in Nelson, 2011), and deposition of the lipophilic
heteropolyester suberin into the parenchymatous bundle
sheath cell wall (Hattersley and Browning, 1981).
Lipophilic cell wall deposits are common to all land plant
lineages, and are thought to have been essential to the transition from aquatic life (Rensing et al., 2008). In grass roots,
suberin is ubiquitously deposited beneath the primary cell
wall in the endodermis (Esau, 1965), and variably in the
root exodermis (Perumalla et al., 1990). In grass leaves,
the vasculature is sheathed by one or two cell layers; the
innermost layer, the mestome sheath (MS), is ubiquitously
suberized (Hattersley and Perry, 1984). Suberin deposition in the outermost layer, the bundle sheath (BS), occurs
primarily in classical phosphoenolpyruvate carboxykinase (PCK) and NADP-ME-type C4 grasses, with the MS
generally absent from the latter (Hattersley and Browning,
1981; Eastman et al., 1988a). Grasses with classical NADmalic enzyme (NAD-ME) C4 anatomy lack a suberized BS,
but non-classical species with large BS surface areas that
are in contact with the mesophyll (M) may be suberized
(Prendergast et al., 1987). Despite this correlation, BS surface area and cell wall suberization seem to be genetically
unlinked (Ohsugi et al., 1997). Variation in the structure
and development of suberized walls occurs both within and
between species.
Suberized vascular sheaths have several putative physiological functions. For example, suberized cell walls potentially mediate vascular fluxes of solutes and photoassimilates
in all grasses, and have also been implicated in abiotic stress
tolerance (Kuo et al., 1974; Griffith et al., 1985). In C4 species, BS suberization may restrict exchange of gases and
photosynthetic intermediates across the BS/M interface
(Laetsch, 1971). However, extensive suberization may also
reduce biomass quality or digestibility, owing to its enrichment in phenylpropanoid precursors (Akin et al., 1983;
Schreiber et al., 1999). Thus, suberized walls are promising
targets for biomass improvement in all grasses, and may be
required to engineer PCK or NADP-ME C4 photosynthesis
into C3 species. Selective manipulation of suberization at the
organ or tissue level is desirable to maximize stress tolerance
and digestibility. Recent studies have indicated an intriguing
molecular link between root endodermal differentiation and
bundle sheath differentiation (Slewinski et al., 2012). Thus,
a deeper understanding of suberin biosynthesis and regulation in leaf tissues may provide an entry into engineering the
pathway in roots as well. However, efforts to dissect vascular
sheath suberization have been confounded by interspecies
variation and a lack of genetic resources in model grasses.
Despite over forty years of research, sheath suberization
remains functionally ambiguous. In this review, we discuss
the development, chemical composition, physiology, and
biosynthesis of suberized cell walls in vascular sheaths of
grass leaves.
Bundle sheath suberization shares
common features with root development
In an early review of root development, van Fleet (1961)
described four states of endodermal cell wall differentiation
common to monocots, including grasses. State I occurs early
in development, when osmiophilic Casparian strips (CS) are
deposited in the radial primary cell walls. CS are tightly associated with the plasmalemma (Esau, 1965), and are comprised
predominately of lignin. Suberin is absent (Naseer et al., 2012)
or a minor component (Zeier et al., 1999; Zeier and Schreiber,
1998). The majority of endodermal suberization occurs during
State II, when suberin lamellae (SL) form beneath the primary
cell wall and surround the symplast (Zeier et al., 1999; Robards
and Robb, 1972). SL are thin (25–40 μm), lamellar, osmiophilic
deposits except near plasmodesmata (PD), where thickened
lamellae constrict the aperture of the pore (Haas and Carothers,
1975). Following SL development, a tertiary wall forms asymmetrically (State III). The tertiary wall is then enriched with
lignin with the thickest region lying on the inner tangential wall
in a characteristic “U-shape” (State IV; van Fleet, 1961). To
date, maize (Zea mays) is the only grass for which tertiary wall
composition has been analysed. The tertiary wall is entirely lignocellulosic, and additional suberin deposition does not occur
at this stage (Zeier et al., 1999). Likewise, in both maize and
non-graminaceous monocots, lipophilic Sudan III staining is
limited to the state I CS and state II SL, suggesting that tertiary
walls are exclusively lignocellulosic across broad taxa (Zeier
and Schreiber, 1998; Zeier et al., 1999).
Although not strictly analogous, grass leaf BS and root endodermal development share several common features. A grass
leaf sampled at the proper developmental state encompasses
a complete sink-to-source gradient, which matures basipetally
(Evert et al., 1996a; Li et al., 2010). States II–IV of root endodermal maturation are common to the leaf vascular sheaths
surrounding large, intermediate, and minor veins (Fig. 1A, B
and Fig. 2A). Although CS form ubiquitously within millimetres of the apex in grass roots (Robards and Robb, 1972; Haas
and Carothers, 1975; Clark and Harris, 1981), they are absent
from both the BS and MS (Eastman et al., 1988a). As in roots,
State II SL are 25–40 μm thick, constrict plasmodesmata, and
can surround all or part of the symplast (Eastman et al., 1988a;
Evert et al., 1977; Robinson-Beers and Evert, 1991a, b). In both
BS and MS, SL may be continuous around the cell periphery,
as in major vein MS cells of rice, oat (Avena byzantia), and barley (Hordeum vulgare) (O’Brien and Carr, 1970; Chonan et al.,
1981; Evert et al., 1996b). Alternatively, they may be discontinuous or absent in the inner tangential wall as in maize BS (Evert
et al., 1977). Suberization may also vary within a single vascular bundle. For example, in sugarcane (Saccharum hybrid), BS
cell walls have a continuous SL when adjacent to xylem and are
limited to the outer tangential and radial walls when adjacent
to phloem (Robinson-Beers and Evert, 1991a). In the majority
of grasses sampled to date, suberization was assayed at a single
time point, and comparatively little is known about suberization in the context of leaf development.
Vascular sheath suberization follows cell elongation, and
may either be synchronous in all vein orders and cell positions
Bundle sheath suberization in grass leaves | 3373
Fig. 1. Ultrastructure of the bundle sheath cell wall. (A) A bundle sheath
cell from a small vascular bundle of the NADP-ME C4 grass maize. A State
II suberin lamella (SL; solid blue line) is deposited in the outer tangential
wall (OTW) and radial wall (RW) along the bundle sheath/mesophyll
interface, but is absent from the inner tangential wall (ITW). State
I Casparian Strips (CS) are present in the root endodermis (not pictured)
but absent from the equivalent positions in the RW (dashed blue box).
Chloroplasts are centrifugally positioned along the OTW. The solid blue
box indicates the area enlarged in Fig. 1B. (B) Fine structure of the bundle
sheath cell wall. The outermost layer is a polysaccharide primary cell wall
(PCW). A State II suberin lamella (SL) is deposited beneath the primary
wall, followed by a State III/IV lignocellulosic tertiary cell wall (TCW). The
plasma membrane (PM) is immediately adjacent to the TCW. Scale bars
denote 10 μm in A and 1 μm in B.
within a bundle as in maize BS (Evert et al., 1996a), or asynchronous as in wheat (Triticum aestivum) MS (O’Brien and
Kuo, 1975). In juvenile maize leaves, BS cells suberize concurrently with metaxylem lignification and chloroplast differentiation after thin-walled sieve tube maturation is complete
(Evert et al., 1996a; Li et al., 2010). Conversely, wheat MS
cells suberize concurrently with adjacent vascular tissue, and
phloem-adjacent cells mature before xylem-adjacent neighbours (O’Brien and Kuo, 1975). In agreement with these
data, candidate suberin-biosynthesis genes reach maximum
expression at the rice leaf base, (Wang et al., unpublished
observations), whereas maize homologues peak in the transitional zone concurrently with lignin biosynthesis (Li et al.,
2010). Leaf developmental profiles from non-NADP-ME
Panicoideae species are needed to determine whether asynchronous suberization is a general feature of MS versus BS
development, or is specific to C3 Pooideae.
Vascular sheath cell wall development is
not strictly analogous to the endodermis
Compared with both root suberization and leaf cuticle formation, which shares common monomer constituents with
suberin synthesis (Pollard et al., 2008), little is known about
Fig. 2. Proposed functions of suberized cell walls in C4 grasses. (A)
Organization of suberized cell layers around large veins of C3 and C4
grasses. In classical NADP-ME and PCK C4 species, suberin lamellae
(black lines) are continuous in outer tangential and radial walls and variable
in inner tangential walls of both vascular sheaths. Both NAD-ME and C3
grasses possess a suberized mestome sheath (MS) and an unsuberized
bundle sheath (BS). The MS is absent in all veins of NADP-ME species
and in intermediate and small veins of other subtypes. Chloroplasts in the
BS are centrifugally oriented only in suberized C4 species (All) and show
variable placement in PCK species. Chloroplasts are centripetally arranged
in NAD-ME grasses and are less abundant in C3 BS (few). BS areas are
not drawn to scale. (B) Large veins of classical PEPCK and non-classical
NAD-ME C4 grasses. Diffusion of C4 acids (C4; green line) and sucrose
(Suc; purple line) synthesized in mesophyll (M) cells into the vascular
bundle is symplastic; ovals indicate passage through plasmodesmata.
Apoplastic diffusion of released CO2 and Suc is prevented at suberized
walls (bent arrows). Likewise, O2 (black line) can diffuse into M but not
BS cells (bent arrow). In this model, sucrose travels symplastically to the
vascular parenchyma (VP), enters the apoplast (dashed purple line), and
is loaded into the sieve tube-companion cell complex (CC & ST). Water
and dissolved solutes (blue line) exit the metaxylem vessel (MX) wall and
travel apoplastically through radial walls between suberin lamellae of
adjacent BS and MS cells. Dashed lines crossing cell walls indicate that
a metabolite crosses the plasma membrane. (C) Intermediate and small
veins of classical PCK and non-classical NAD-ME C4 grasses, and all veins
of classical NADP-ME C4 grasses. Cell wall ultrastructure and metabolite
transport occur as described in A., but the MS is discontinuous (not
pictured) or absent.
the developmental plasticity of vascular sheath suberization.
Root suberization is highly plastic. For instance, maize State
II SL are developmentally delayed in both endo- and exodermis in hydroponically grown roots relative to aeroponics and
vermiculite (Zimmermann et al., 2000; Enstone and Peterson,
2005). Likewise, cuticular wax deposition starts before the
emergence of a developing leaf from the ligule of its predecessor, and can be induced in younger, elongating cells near
the leaf base by manual peeling of the ligule (Richardson
et al., 2005). Conversely, cutin biosynthesis occurs constitutively in the region of cell elongation at the emerging leaf base
and deposition does not continually increase in parallel with
wax synthesis (Richardson et al., 2007). At least in maize, BS
suberization also seems to be constitutive; despite variation
in growth conditions (irradiance, temperature, and photoperiod), plant age, leaf sampled, and degree of leaf emergence
from the ligule, suberization occurred at identical developmental states in diverse inbreds B73 and W273 (Evert et al.,
1996a; Li et al., 2010).
3374 | Mertz and Brutnell
As discussed above for State II SL, the rate of State III/
IV tertiary wall maturation varies greatly by root order
and environment (Robards and Robb, 1972; Enstone and
Peterson, 2005), but is not well characterized in leaves. Both
BS and MS possess lignocellulosic tertiary walls, but endodermis-like asymmetrical thickenings are present only in
the MS (Eastman et al., 1988a; Evert et al., 1977; O’Brien
and Kuo, 1975). However, in maize, cell identity of both
endodermis and BS are regulated by a common Scarecrow
(ZmSCR) homologue (Slewinski et al., 2012). Thus, divergent wall architectures can occur downstream of cell identity regulators. The molecular mechanism of asymmetric cell
wall deposition is unknown for both SL and tertiary walls.
For tertiary walls, cellulose deposition regulated by localized microtubule depolymerization may lead to patterns of
asymmetrical thickening, as demonstrated for xylem vessels
(Oda et al., 2010; Pesquet et al., 2010). Similarly, localized
lignin polymerization domains, as recently reported for
Arabidopsis thaliana endodermal CS (Roppolo et al., 2011;
Lee et al., 2013), may define sites of lignification. Whether
a similar system occurs in monocots remains an open
question.
Suberin composition varies qualitatively
and quantitatively between species
and organs
The developmental heterogeneity of suberin biosynthesis
described above is accompanied by considerable compositional variation within the SL themselves. Suberin is a heterogeneous polyester matrix comprised of acyl lipid-derived
aliphatic and phenylpropanoid-derived aromatic components (reviewed in Pollard et al., 2008). Suberin shares many
common monomers with epidermal cutin (Fig. 3); both are
lipophilic cell wall matrices of glycerol and oxidized longchain (16:0, 18:0, and 18:1) fatty acids (LCFA), but suberin
is enriched in aromatics and very long-chain (≥20:0) aliphatic
monomers (VLCFA; reviewed by Pollard et al., 2008).
Much remains to be learned about the variation in grass
suberin composition between species and organs, particularly
in the vascular sheaths. In roots, suberin varies both qualitatively and quantitatively between species. For instance,
maize endodermal suberin is 34-times less abundant per unit
area than rice, but is synthesized from a more diverse array
of VLCFA (Schreiber et al., 2005a). Aliphatic composition
Fig. 3. A simplified pathway of grass aliphatic suberin biosynthesis modelled on Arabidopsis thaliana. Following de novo fatty acid synthesis in
the plastid, long-chain fatty acid (LCFA) precursors are converted to acyl-CoA thioesters by a long-chain acyl-CoA synthetase (LACS). Within the
endoplasmic reticulum (ER), LCFA are oxidized to omega-hydroxy or dihydroxy acids (ω-OH, di-OH) by cytochrome P450s (CYP). ω-OH LCFA may be
oxidized by an unknown enzyme to produce dicarboxylic acids (DCA). Before oxidation, LCFA may be elongated to very long-chain fatty acids (VLCFA)
by the fatty acid elongase (FAE) complex. The FAE is comprised of a ketoacyl-CoA synthase (KCS), a ketoacyl-CoA reductase (KCR), a hydroxyacylCoA dehydrase, and an enoyl-CoA reductase (ECR). Both LCFA and VLCFA can be reduced to primary alcohols by fatty acyl-CoA reductase (FAR) or
esterified to glycerol-3-phosphate (G3P) by glycerol-3-phosphate acyltransferase (GPAT) to produce sn-2 monoacylglycerol (2-MAG). In the cytosol,
aliphatic suberin feruloyl transferase (ASFT) can esterify the monolignol precursor ferulic acid to ω-OH fatty acids and primary alcohols of various
chain lengths to form alkyl ferulates. Monomers reach the plasma membrane by an unknown mechanism and are putatively exported by ATP-binding
cassette subfamily G (ABCG) members. Polyesters may be synthesized by a GDSL-like lipase/acylhydrolase (GDSL). Enzymes denoted with an asterisk
are inferred from cutin synthesis. Grass suberin candidates discussed in the text are highlighted in red. Green frames denote monomers abundant
in both suberin and cutin, whereas blue frames denote monomers that are suberin-enriched. Values in parentheses denote numbers of putative
maize homologues for each Arabidopsis protein (Wang et al., unpublished observations). The two groups of ABCG candidates denote half and whole
transporters, respectively (Verrier et al., 2008). Putative GPAT5 substrate specificities were inferred from (Yang et al., 2012).
Bundle sheath suberization in grass leaves | 3375
also varies within a species by tissue type and developmental age. In State I CS, LCFA predominate in both sorghum
(Sorghum bicolor) and maize (Zeier et al., 1999; Espelie and
Kolattukudy, 1979a), whereas omega-hydroxy fatty acids predominate in State II SL (Zeier et al., 1999; Schreiber et al.,
2005a; Soukup et al., 2007). Generally, VLCFA content
increases proportionally with tissue age (Zeier et al., 1999;
Soukup et al., 2007).
To date, vascular sheath suberin content has been profiled only in maize BS and rye (Secale cereale) MS (Griffith
et al., 1985; Espelie and Kolattukudy, 1979b). As expected
for State II SL, both tissues contain significant amounts of
omega-hydroxy fatty acids. In the maize BS, LCFA predominate over VLCFA; thus, mature SL in BS and endodermis
have similar chain length distributions of monomers (Zeier
et al., 1999; Espelie and Kolattukudy, 1979b). However, polyhydroxy and epoxy fatty acids are the dominant constituent
of BS and MS, respectively (Griffith et al., 1985; Espelie and
Kolattukudy, 1979b). Polyhydroxy fatty acids are a major
constituent of leaf cutin in many species (reviewed by Pollard
et al., 2008). Both monomers are also major components of
leaf cutin in these grasses; thus, vascular sheath suberin composition shares similarities with epidermal cutin as well as
root suberin. The functional implications of variable suberin
composition are unclear.
Like aliphatic suberin, aromatic suberin varies quantitatively between species and generally increases with tissue age (Schreiber et al., 2005a; Soukup et al., 2007). The
monolignol precursors coumarate and ferulate comprise the
majority of aromatic suberin in all grasses studied to date
(Zeier et al., 1999; Schreiber et al., 2005a; Soukup et al.,
2007). However, accurate quantification of aromatic suberin
in grasses is complicated by extensive esterification of ferulate and coumarate to arabinoxylans in the polysaccharide
cell wall (Schreiber et al., 2005a; Harris and Hartley, 1976;
Mueller-Harvey et al., 1986). These ferulate monomers,
like monolignols, can undergo oxidative coupling to form
covalent crosslinks to each other and to lignin (reviewed by
Ralph et al., 2004; Ralph, 2010). Although coumarate does
not dimerize in planta (Ralph et al., 1994), it is thought to
function as a nucleation site for polymerization of other
monolignols (reviewed in Ralph, 2010). Thus, phenolic
suberin in vascular sheath cell walls is ideally positioned to
crosslink with both hemicellulose and lignin and may contribute to biomass recalcitrance. This is especially true for
C4 species, where veins occupy the largest proportion of leaf
tissue area (Hattersley, 1984). Accordingly, suberized portions of the BS and MS cell walls are recalcitrant to degradation in rumen fluid relative to parenchymatous C3 BS
cells with polysaccharide primary walls (Akin et al., 1983).
However, unsuberized C4 NAD-ME BS cells are the least
digestible, suggesting that suberized cell walls are less recalcitrant than lignocellulosic walls (Wilson and Hattersley,
1983). As discussed above, although aromatic suberin is necessary for barrier function in model dicots (see Ranathunge
et al., 2011, and references therein), the precise relationship between monomer content and physiological function
remains an open question.
Vascular sheath suberization may affect
multiple aspects of leaf physiology
Although vascular sheath suberization has been correlated
with multiple facets of leaf physiology (Fig. 2B, C), causal
relationships have not been established. O’Brien and Carr
(1970) proposed that suberin lamellae restrict passive loss of
water and solutes from the vasculature in both BS and MS
cells. BS cells resist plasmolysis when exposed to concentrated
(1.5 M) sucrose (Evert et al., 1978) and desiccation stress
(Giles et al., 1974; Giles et al., 1976), indicating low apoplastic permeability. Accordingly, ions dissolved in the transpiration stream can enter the tertiary walls of BS cells, but
cannot diffuse across the SL to the primary cell wall (Fig. 1B
and Fig. 2B, C) (Botha et al., 1982; Evert et al., 1985). SL in
radial walls of adjacent MS/BS cells do not fuse; thus, water
and solutes diffuse through the radial walls of all vein orders
in both C3 and C4 grasses (Evert et al., 1985; Peterson et al.,
1985; Canny, 1986; Eastman et al., 1988b). As the CS are the
major endodermal permeability barriers in radial walls, the
absence of these structures in vascular sheaths may explain
their permeability to ions (Peterson et al., 1993).
Likewise, BS and MS suberization may restrict apoplastic backflow of sucrose out of the vascular bundle, facilitating spatial separation of transpirational efflux and phloem
loading (Kuo et al., 1974; Canny, 1986). In the majority of
grasses studied to date, sucrose synthesized in M cells travels
via PD to vascular parenchyma cells, where it enters the apoplast for uptake by companion cells (Fig. 2B, C) (reviewed
in Braun and Slewinski, 2009). Accordingly, PD density in
these species is highest along a symplastic pathway leading
from M cells through BS, MS (if present), and into vascular
parenchyma (VP) cells, the site of sucrose entry into the apoplast (Robinson-Beers and Evert, 1991b; Evert et al., 1996b;
Evert et al., 1978; Botha, 1992). PD frequency is highest in
small veins and lowest in large veins, consistent with functional specialization for phloem loading by small veins (Fritz
et al., 1989). Patterns of suberization are consistent with a
role for SL in restricting sucrose diffusion to a symplastic
route. During tissue maturation, vascular sheath suberization
is completed before the sink–source transition (Evert et al.,
1996a). Likewise, when MS suberization is asynchronous
within individual bundles, complete SL appear first adjacent to phloem cells (O’Brien and Kuo, 1975). Furthermore,
although SL are discontinuous in the inner tangential walls
bordering phloem-associated vascular parenchyma in many
PCK and NADP-ME C4 species, they are present near PD
(Evert et al., 1977; Robinson-Beers and Evert, 1991a). The
absence of SL in portions of these walls may provide an
apoplastic route into the vasculature. However, the bypass
does not occur in the maize sucrose export defective1 (sxd1)
mutant, in which BS/VP PD are occluded by callose (Russin
et al., 1996; Botha et al., 2000). This suggests that other cell
wall components, potentially lignin, may also contribute significantly to diffusion resistance.
In addition to the proposed roles for SL in all grasses,
specialized functions may occur in PCK or NADP-ME C4
species. For example, suberization along the entire C4 BS/M
3376 | Mertz and Brutnell
interface is hypothesized to restrict photosynthetic intermediates to a symplastic route (Evert et al., 1977). As PD are the
primary limiting factor for metabolite diffusion, their abundance correlates with net C4 photosynthetic rate, and PD frequency at the BS/M interface is significantly higher in all C4
subtypes relative to C3 species (Botha, 1992). Accordingly,
although isolated BS strands accumulate metabolites, neither
plasmolysed BS strands nor protoplasts can do so (Weiner
et al., 1988). Although recent models of C4 metabolite
exchange include suberized PD constrictions as a limiting
factor (Sowinski et al., 2008), the functional impact of these
structures remains unclear. For instance, the molecular weight
size exclusion limit of BS PD (approximately 850) is comparable between classical PCK and NAD-ME species; thus, the
plasmodesmatal neck constriction common to both groups,
and not the SL may be the primary determinant of size exclusion (Weiner et al., 1988). Likewise, PD abundance per unit
vein at the BS/M interface is highest in the NAD-ME type
(Botha, 1992). This suggests that metabolite flux is mostly or
entirely symplastic even in the absence of SL.
Laetsch (1971) proposed that BS SL are barriers that prevent apoplastic diffusion of CO2 and O2 across the BS/M
interface (Laetsch, 1971). Suberized tissues have low diffusional O2 permeability (Ranathunge et al., 2003) and regulate
gas exchange in root exodermis, periderm and lenticels, and
root nodules of legumes (Jacobsen et al., 1998; Lendzian,
2006; Kotula et al., 2009). Likewise, the CO2 permeability of
C4 BS cells is over 100-fold lower than C3 M cells (Furbank
et al., 1989). Unsuberized NAD-ME species also exhibit low
permeability; thus, BS permeability results from a combination of apoplastic modifications and cellular metabolism,
and SL are not obligatory for CO2 concentration (Jenkins
et al., 1989; von Caemmerer and Furbank, 2003). However,
NAD-ME grasses exhibit a suite of anatomical modifications
including lignocellulosic secondary walls, centripetal chloroplast orientation, and a decreased BS surface area:volume
ratio that may limit gas exchange (Hattersley and Browning,
1981). Centrifugally oriented chloroplasts tightly associated
with the BS/M interface are common only in species with a
suberized BS (Hattersley and Browning, 1981; Hatch et al.,
1975). Interestingly, BS suberization and chloroplast orientation segregate independently in interspecific Panicum hybrids
(Ohsugi et al., 1997). Hybrid plants were suberized with variable BS chloroplast orientation. Dry matter carbon isotope
ratios (δ13C) were identical in the hybrid lines and higher
than in classical NAD-ME species, suggesting that SL contribute to a lower (more C4-like) degree of carbon isotope
discrimination in closely related species (Ohsugi et al., 1997).
However, the validity of dry matter carbon isotope ratios as
a measure of CO2 leakage from BS strands remains tenuous
owing to confounding variation from plant metabolism (von
Caemmerer and Furbank, 2003, and references therein).
Stress physiology of suberized vascular sheaths is largely
uncharacterized. Both total MS suberization and the proportion of epoxy fatty acids increased during cold acclimation
of rye (Griffith et al., 1985), but the molecular mechanism
by which MS suberization promotes cold tolerance remains
ambiguous. Conversely, short-term ultrastructural changes in
BS suberization were not reported during drought stress and
subsequent rehydration of either maize or sorghum (Giles
et al., 1974; Giles et al., 1976). Additional studies of vascular sheath suberization under both biotic and abiotic stress
are needed in grasses, both to expand existing knowledge of
barrier function, and to facilitate candidate gene identification. For example, two candidate suberin-biosynthesis genes
were recently identified in rice roots undergoing salt-induced
suberin deposition (Krishnamurthy et al., 2009).
Molecular genetic dissection of
biosynthesis and regulation
Mutants with altered suberization are necessary to assess
the functions of SL in a common genetic background with
minimal confounding variation. Despite substantial progress toward elucidating the molecular genetic basis of both
suberin and cutin biosynthesis in the C3 dicots Arabidopsis
and potato (Solanum tuberosum; reviewed by Ranathunge
et al., 2011; Beisson et al., 2012; Yeats and Rose, 2013), no
suberin biosynthesis genes have been characterized in any
grass species. However, biochemical and forward genetics
approaches have identified several candidate genes potentially involved in suberin biosynthesis (Fig.3). Although these
candidates have been studied exclusively in roots, tissue-specific transcript profiling indicates that many putative suberinbiosynthesis genes are expressed in both roots and leaves
(Sekhon et al., 2011).
cDNA isolation from maize roots yielded a 3-ketoacylCoA synthase (KCS), which is homologous to AtKCS1 (Todd
et al., 1999; Schreiber, 2000), and a putative suberin-associated O-methyltransferase, Zea Root Preferential 4 (ZmZPR4)
(Held et al., 1993). Biochemical evidence of anionic peroxidase and fatty acid elongase (FAE) complex activity in maize
seminal roots undergoing endo- and exodermal suberization was also reported (Pozuelo et al., 1984; Schreiber et al.,
2005b). In the latter case, the chain-length specificity was consistent with aliphatic suberin monomer content, supporting
the role of the KCS in suberin biosynthesis (Schreiber et al.,
2005b). KCS is associated with a FAE complex involved in
sequential acyl chain elongation (Joubes et al., 2008). The
cuticular wax-associated 3-ketoacyl-CoA reductases (KCRs)
GLOSSY8A and GLOSSY8B are also components of the
maize FAE complex, and thus may contribute to suberin
biosynthesis (Xu et al., 1997; Xu et al., 2002; Dietrich et al.,
2005). Therefore, a detailed evaluation of wax-biosynthesis
mutants for pleiotropic suberin phenotypes may identify
suberin-biosynthesis genes.
Bifunctional Arabidopsis KCS that contribute to both
suberin and wax biosynthesis exist (Franke et al., 2009; Lee
et al., 2009), as do ATP-binding cassette subfamily G transporters involved in monomer secretion to the apoplast (Bird
et al., 2007; Panikashvili et al., 2010). Recently, a rice KCS with
cuticular wax defects and ubiquitous expression was characterized and proposed to contribute to multiple cellular processes
requiring the FAE complex, including suberin biosynthesis (Yu
et al., 2008). Likewise, two homologous ATP-binding cassette
Bundle sheath suberization in grass leaves | 3377
subfamily G transporters from rice and barley exhibit cuticular
wax defects and impaired transpiration barrier function, and
are expressed throughout the leaf elongation zone, including
the MS (Chen et al., 2011). Thus, multifunctional wax-biosynthesis genes probably exist in grasses as well. However, a
second rice KCS shows an epidermal localization consistent
with a specific function in cuticular wax biosynthesis (Ito et al.,
2011). Therefore, tissue-specific expression data is needed to
refine the selection of suberin biosynthesis candidates, particularly for large, redundant gene families.
Delineating suberin-associated KCS genes is also of interest because these enzymes are thought to be rate limiting for
VLCFA biosynthesis (Millar and Kunst, 1997). Thus, mining
their promoter regions for cis-regulatory elements may elucidate transcriptional regulators of suberin biosynthesis, which
are uncharacterized in any species.
It was recently reported that in oat addition lines containing maize chromosome 3, lipophilic material is ectopically deposited in C3 BS cell walls (Tolley et al., 2012). The
deposition pattern resembles the maize BS; however, SL are
absent, and the authors concluded that additional loci are
required for their biosynthesis (Tolley et al., 2012). A homologue of the secondary cell wall regulator SECONDARY
WALL-ASSOCIATED NAC DOMAIN 2 (AtSND2) is
among the candidate loci on chromosome 3 (Li et al., 2010;
Tolley et al., 2012). Recently, a homologue of the Arabidopsis
SCARECROW (AtSCR) transcription factor was also shown
to affect endodermal CS formation and BS suberization in
maize (Slewinski et al., 2012; DiLaurenzio et al., 1996). This
study supports developmental similarities of vascular sheaths
between organs, but the precise effect of SCR on suberin biosynthesis remains unclear.
Conclusions and future directions
Suberized cell layers surrounding the vasculature are a ubiquitous feature of grass leaves. However, variation in development
and monomer content exists both within and between species,
with unclear effects on barrier function. Sheath suberization
has been implicated in numerous physiological processes, but
interspecies variation has complicated efforts to dissect barrier function. Mutants with altered suberization are needed to
characterize BS function in the grasses. The genomic resources
(Draper et al., 2001; Matsumoto et al., 2005; Paterson et al.,
2009; Schnable et al., 2009; Bennetzen et al., 2012), and expression data (Li et al., 2010; Sekhon et al., 2011; Wang et al.,
unpublished observations) needed to facilitate candidate identification are available, and a putative biosynthesis pathway has
been partially delineated (Fig. 3; Li et al., 2010; Wang et al.,
unpublished observations). Both stable mutants and transgenic lines can now be generated in both C3 and C4 model
grasses (Draper et al., 2001; An et al., 2005; Brutnell et al.,
2010; Vollbrecht et al., 2010; Bragg et al., 2012). For example, to investigate whether BS suberization forms an essential
gas exchange barrier for NADP-ME C4 photosynthesis, we
are targeting several VLCFA-modifying candidate genes in
maize and green millet (Setaria viridis). We recently mutated
putative homologues of Arabidopsis ALIPHATIC SUBERIN
FERULOYL TRANSFERASE (AtASFT; Gou et al., 2009;
Molina et al., 2009) using Ds transposons in maize and monocot-optimized RNAi vectors in millet (Vollbrecht et al., 2010;
Mann et al., 2012; Mertz et al., unpublished observations).
Resources for PCK C4 grasses are currently limited, but a
similar targeted reverse genetics approach is feasible for both
NAD-ME C4 (Switchgrass, Panicum virgatum) and C3 (rice
or Brachypodium distachyon) model grasses. Thus, after forty
years of ambiguity, the stage is now set for a rapid expansion
of our understanding of sheath suberization in grasses.
Acknowledgements
We thank Richard Medville and Robert Turgeon (Cornell University) for
kindly providing the EM image shown in Fig. 1. We also wish to thank two
anonymous reviewers for their constructive comments. This work was supported by an NSF grant to TPB. (IOS- 1127017).
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